In phase contrast microscopy, an object is illuminated by an illumination optical system defined by an aperture stop located at the pupil position of the illumination optical system. A phase plate, providing a phase modulation, is arranged inside an objective lens at a location conjugate with the pupil of the illumination optical system. Then, the phase difference of light introduced by the object (phase object) is converted into a difference in light intensity. Therefore, the phase difference of the object is visualized as light and shade of the image (i.e., contrast), and can be observed. The phase contrast microscope was invented by Fritz Zernike in 1935, and is described in chapter 8.6 of the textbook "Principles of Optics (Sixth Edition)," by M. Born and E. Wolf, Pergamon Press, 1980.
The principle of the phase contrast microscope will be explained. With reference to FIG. 1, prior art phase contrast microscope 10 comprises, in order along an optical axis AX, a light source LS capable of emitting light L, an aperture stop AP arranged at a pupil plane P1 and having an annular opening (aperture) AO (FIG. 2), a condenser lens G1 having a front focal point F positioned on-axis at pupil plane P1, an object stage OS for supporting an object O to be observed, a first objective lens G2 having a rear focal point F' positioned on-axis, a phase plate Ph0 arranged at rear focal point F' and optically conjugate to aperture AP (FIG. 3), a second objective lens G3, and an image plane IP. First and second objective lenses G2 and G3 constitute an objective optical system. Phase plate Ph0 has an opening (aperture) AO0 similar to aperture opening AO, and also has a phase film PP thereon covering annular opening AO0. Phase film PP provides a phase difference of a quarter wavelength to light transmitted therethrough. Also, phase plate Ph0 has the same shape as phase film PP, and has an absorbing film which reduces the amount of transmitted light.
The operation of phase contrast microscope 10 is now explained. Illumination light L of wavelength .lambda. is emitted from light source LS and passes through annular opening AO in ring aperture AP. The latter determines the amount and nature of llumination of object O. Objective lenses G2 and G3 collect the light transmitted through object O and form an image of the object on object plane IP.
Light L is diffracted upon passing through object O and is thereby separated into a direct (undiffracted) light beam L1 and a .+-.1.sup.st order diffracted light beam L2. Light beams L1 and L2 then pass through phase plate Ph0. Phase film PP covers aperture opening AOD, which changes the phase of light beam L1 by a quarter of the wavelength (.lambda./4) relative to diffracted light beam L2. Light beam L1, with its phase advanced by (.lambda./4) interferes destructively with diffracted light beam L2 at image plane IP. On the other hand, light L having passed through a part of object O having no phase-altering properties does not produce diffracted light, and takes part in the background of direct light beam L1. Therefore, the phase difference of object O can be observed as light and shade in the image. Moreover, the amplitude of .+-.1.sup.st order diffracted light L2 can be expressed as a Bessel function J.sub.1 (B). According to the intensity J.sub.1 (B).sup.2 of the diffracted light varying in accordance with the amount of the phase difference, a transmittance-modulation film, such as a neutral density film ND reducing the amount of transmitted light, is applied to the phase film PP. If the amplitude of direct light beam L1 is made equal to that of the diffracted light beam L2 with the help of neutral density transmittance-modulation film ND, the phase object can be observed with maximum intensity contrast against the background.
According to prior art contrast microscope 10 described above, if the amount of the phase difference of the object is small, a high detection sensitivity to the amount of phase difference, using low transmittance of direct light beam L1, is utilized. This is referred to as a high-contrast type microscope, or high contrast imaging. In this instance, the transmittance of the modulation film applied on phase film PP is about 0.1 to 0.25. In the case of a high-contrast imaging, an object having a small amount of phase difference can be easily observed. However, when an object having large amount of phase difference is observed, the ratio of the amplitude of the direct light to that of the diffracted light is reversed, and a reverse contrast image is formed. Therefor, a fringe-shaped blurring of light in accordance with the phase difference or structure of the object is formed around the image of the object. The phenomenon is called "halo." If halo is produced, good observation of the object is disturbed. Moreover, there is good possibility of misidentification of structure in the object.
When an object having a large amount of phase difference is observed, a low-contrast type phase contrast microscope having high transmittance of the direct (undiffracted) light beam is utilized to avoid producing halo. In such a microscope, it is desirable that the transmittance of transmittance-modulation film ND applied on phase film PP is on the order of 0.25 to 0.50. In a low-contrast type phase contrast microscope, instead of producing halo, other problems occurs. For example, when observing an object having small amount of phase difference, it is difficult to get a good image because of low image contrast.
In addition to the problems mentioned above, when an object is observed with white light using a phase contrast microscope, further problems arise. For example, with reference to FIG. 4, consider a phase object 11 having an interior medium width t and a refractive index n1, surrounded by a medium of refractive index n2. When phase object 11 is observed with a phase-contrast microscope, although refractive index n1 varies in accordance with wavelength, the refractive index is considered to be approximately constant within a normal extent of dispersion. The optical path length inside of phase object 11 can be expressed as n1.times.t. The amount of phase difference is expressed as (n1-n2).times.t. Since the amount of phase difference is expressed in units of wavelength, the amount of phase difference produced in the same object is approximately inversely proportional to the wavelength. For example, an object having the amount of phase difference of 0.1.lambda. at a wavelength of 550 nm will produce the amount of phase difference of 0.14.lambda. at a wavelength of 400 nm. Therefore, there is a problem that even an object producing no halo at a given wavelength, such as 550 nm, produces halo at a different wavelength, such as 400 nm.
To solve this problem, the transmittance T1 of transmittance-modulation film ND is varied in accordance with the wavelength, from about 0.1 to about 0.4. For example, with reference also to FIG. 5, the transmittance T.sub.ND of the transmittance-modulation film ND at short wavelengths is made higher than that at long wavelengths. This allows the phase contrast microscope to work as low-contrast type at short-wavelengths and as high-contrast type at long wavelengths. Therefore, it is possible to mitigate the generation of a halo likely to be produced at the short-wavelength side. However, if the transmittance of the transmittance-modulation film ND is set high at short wavelengths, the spectral transmittance of the background of the image is changed, so that the background has color. For example, if the transmittance is high at short wavelengths, as shown in FIG. 5, the background becomes blue.